Generation of plants with improved drought tolerance

ABSTRACT

The present invention is directed to plants that display a drought tolerance phenotype due to altered expression of a DR05 nucleic acid. The invention is further directed to methods of generating plants with a drought tolerance phenotype.

REFERENCE TO RELATED APPLICATIONS

This application is the §371 U.S. National Stage of InternationalApplication No. PCT/US2004/20323, filed Jun. 23, 2004, which waspublished in English under PCT Article 21(2), which in turn claims thebenefit of U.S. provisional patent application 60/482,075, filed Jun.24, 2003, the contents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Crop production is affected by numerous abiotic environmental factors,with soil salinity and drought having the most detrimental effects.Approximately 70% of the genetic yield potential in major crops is lostdue to abiotic stresses, and most major agricultural crops aresusceptible to drought stress. Attempts to improve yield under stressconditions by plant breeding have been largely unsuccessful, primarilydue to the multigenic origin of the adaptive responses (Barkla et al.1999, Adv Exp Med Biol 464:77-89).

Considerable effort has focused on the identification of genetic factorsthat contribute to stress tolerance and on the genetic engineering ofcrop plants with increased stress tolerance. A number of genes have beenidentified whose expression or mis-expression is associated with droughttolerance, via a variety of different mechanisms. For instance,transformed tobacco that express maize NADP-malic enzyme displayincreased water conservation and gained more mass per water consumedthan wild-type plants (Laporte et al. 2002, J Exp Bot 53:699-705).Significant research effort has focused on the plant hormone abscisicacid (ABA), which is involved in adaptation to various environmentalstresses. Transgenic tobacco and transgenic Arabidopsis that overexpressthe enzyme 9-cis-epoxycarotenoid dioxygenase (NCED), which is key to ABAbiosynthesis, display improved drought tolerance (Qin et al. 2002, PlantPhysiol 128:544-51; Iuchi et al. 2001, Plant J 27:325-33). Droughttolerance is often linked to salt tolerance, since both are associatedwith regulation of osmotic potential and turgor. Accordingly, transgenicplants that overexpress a vacuolar H+ pump (H+-pyrophosphatase), whichgenerates a proton gradient across the vacuolar membrane, displayimproved drought- and salt-stress, due to increased solute accumulationand water retention (Gaxiola et al. 2001, Proc Natl Acad Sci USA98:11444-9). Trehalose also contributes to osmoprotection againstenvironmental stress. Potato plants the mis-expresstrehalose-6-phosphate synthase, a key enzyme for trehalose biosynthesis,show increased drought tolerance (Yeo et al. 2000, Mol Cells 10:263-8).

Arabidopsis has served as a model system for the identification of genesthat contribute to drought tolerance. For instance, researchers haveidentified numerous genes that are induced in response to waterdeprivation (e.g., Taji et al. 1999, Plant Cell Physiol 40:119-23;Ascenzi et al., 1997, Plant Mol Biol 34:629-41; Gosti et al. 1995, MolGen Genet 246:10-18; Koizumi et al. 1993 Gene 129:175-82) and cis-actingDNA sequences called ABA responsive elements (ABREs) that control ABA orstress responsive gene expression (Giraudat et al. 1994, Plant Mol. Biol26: 1557).

Several drought tolerant mutants of Arabidopsis have been identified.These include the recessive mutants abh1 (Hugouvieux et al. 2001, Cell106: 477), era1-2 (Pei et al. 1998, Science 282: 286) and abi1-1Ri(Gosti et al. 1999, Plant Cell 11:1897-1909). The mutants era1-2 andabh1 were identified by screening for seedlings hypersensitive to ABA,while the mutant abi1-1Ri was isolated as an intragenic suppressor ofthe ABA insensitive mutant abi1-1. Dominant drought tolerant mutantswere identified by over-expressing ABF3, ABF4 (Kang et al. 2002, PlantCell 14:343-357) or DREB1A (Kasuga, 1999 Nature Biotech 17: 287). ABF3and ABF4 encode basic-region leucine zipper (bZIP) DNA binding proteinsthat bind specifically ABREs. DREB1A encodes a protein with an EREBP/AP2DNA binding domain that binds to the dehydration-responsive element(DRE) essential for dehydration responsive gene expression (Liu et al.1998, Plant Cell 10: 1391). A dominant drought tolerant phenotype intobacco was obtained by over-expressing the soybean BiP gene (Alvim etal. 2001, Plant Physiol 126, 1042).

Activation tagging in plants refers to a method of generating randommutations by insertion of a heterologous nucleic acid constructcomprising regulatory sequences (e.g., an enhancer) into a plant genome.The regulatory sequences can act to enhance transcription of one or morenative plant genes; accordingly, activation tagging is a fruitful methodfor generating gain-of-function, generally dominant mutants (see, e.g.,Hayashi H et al., Science (1992) 258: 1350-1353; Weigel D, et al., PlantPhysiology (2000) 122:1003-1013). The inserted construct provides amolecular tag for rapid identification of the native plant whosemis-expression causes the mutant phenotype. Activation tagging may alsocause loss-of-function phenotypes. The insertion may result indisruption of a native plant gene, in which case the phenotype isgenerally recessive.

Activation tagging has been used in various species, including tobaccoand Arabidopsis, to identify many different kinds of mutant phenotypesand the genes associated with these phenotypes (Wilson K et al., PlantCell (1996) 8: 659-671, Schaffer R, et al., Cell (1998) 93: 1219-1229,Fridborg I et al., Plant Cell 11: 1019-1032, 1999; Kardailsky I et al.,Science (1999) 286: 1962-1965; Christensen S et al., 9^(th)International Conference on Arabidopsis Research. Univ. ofWisconsin-Madison, Jun. 24-28, 1998. Abstract 165).

SUMMARY OF THE INVENTION

The invention provides a transgenic plant comprising a planttransformation vector comprising a nucleotide sequence that encodes oris complementary to a sequence that encodes a DRO5 polypeptide or anortholog thereof. The transgenic plant is characterized by havingincreased drought tolerance relative to control plants.

The invention further provides a method of producing an improved droughttolerance phenotype in a plant. The method comprises introducing intoplant progenitor cells a vector comprising a nucleotide sequence thatencodes or is complementary to a sequence encoding a DRO5 polypeptide orortholog thereof and growing a transgenic plant that expresses thenucleotide sequence. In one embodiment, the DRO5 polypeptide has atleast 50% sequence identity to the amino acid sequence presented in SEQID NO:2 and comprises a DUF260 domain (PF03195). In other embodiments,the DRO5 polypeptide has at least 80% or 90% sequence identity to or hasthe amino acid sequence presented in SEQ ID NO:2.

The invention further provides plants and plant parts obtained by themethods described herein.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise indicated, all technical and scientific terms usedherein have the same meaning as they would to one skilled in the art ofthe present invention. Practitioners are particularly directed toSambrook et al. Molecular Cloning: A Laboratory Manual (Second Edition),Cold Spring Harbor Press, Plainview, N.Y., 1989, and Ausubel F M et al.Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y., 1993, for definitions and terms of the art. It is to be understoodthat this invention is not limited to the particular methodology,protocols, and reagents described, as these may vary.

As used herein, the term “vector” refers to a nucleic acid constructdesigned for transfer between different host cells. An “expressionvector” refers to a vector that has the ability to incorporate andexpress heterologous DNA fragments in a foreign cell. Many prokaryoticand eukaryotic expression vectors are commercially available. Selectionof appropriate expression vectors is within the knowledge of thosehaving skill in the art.

A “heterologous” nucleic acid construct or sequence has a portion of thesequence that is not native to the plant cell in which it is expressed.Heterologous, with respect to a control sequence refers to a controlsequence (i.e. promoter or enhancer) that does not function in nature toregulate the same gene the expression of which it is currentlyregulating. Generally, heterologous nucleic acid sequences are notendogenous to the cell or part of the genome in which they are present,and have been added to the cell, by infection, transfection,microinjection, electroporation, or the like. A “heterologous” nucleicacid construct may contain a control sequence/DNA coding sequencecombination that is the same as, or different from a controlsequence/DNA coding sequence combination found in the native plant.

As used herein, the term “gene” means the segment of DNA involved inproducing a polypeptide chain, which may or may not include regionspreceding and following the coding region, e.g. 5′ untranslated (5′ UTR)or “leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons) and non-transcribed regulatory sequence.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention.

As used herein, the term “gene expression” refers to the process bywhich a polypeptide is produced based on the nucleic acid sequence of agene. The process includes both transcription and translation;accordingly, “expression” may refer to either a polynucleotide orpolypeptide sequence, or both. Sometimes, expression of a polynucleotidesequence will not lead to protein translation. “Over-expression” refersto increased expression of a polynucleotide and/or polypeptide sequencerelative to its expression in a wild-type (or other reference [e.g.,non-transgenic]) plant and may relate to a naturally-occurring ornon-naturally occurring sequence. “Ectopic expression” refers toexpression at a time, place, and/or increased level that does notnaturally occur in the non-altered or wild-type plant.“Under-expression” refers to decreased expression of a polynucleotideand/or polypeptide sequence, generally of an endogenous gene, relativeto its expression in a wild-type plant. The terms “mis-expression” and“altered expression” encompass over-expression, under-expression, andectopic expression.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell where the nucleicacid sequence may be incorporated into the genome of the cell (forexample, chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (for example,transfected mRNA).

As used herein, a “plant cell” refers to any cell derived from a plant,including cells from undifferentiated tissue (e.g., callus) as well asplant seeds, pollen, progagules and embryos.

As used herein, the terms “native” and “wild-type” relative to a givenplant trait or phenotype refers to the form in which that trait orphenotype is found in the same variety of plant in nature.

As used herein, the term “modified” regarding a plant trait, refers to achange in the phenotype of a transgenic plant relative to the similarnon-transgenic plant. An “interesting phenotype (trait)” with referenceto a transgenic plant refers to an observable or measurable phenotypedemonstrated by a T1 and/or subsequent generation plant, which is notdisplayed by the corresponding non-transgenic (i.e., a genotypicallysimilar plant that has been raised or assayed under similar conditions).An interesting phenotype may represent an improvement in the plant ormay provide a means to produce improvements in other plants. An“improvement” is a feature that may enhance the utility of a plantspecies or variety by providing the plant with a unique and/or novelquality.

An “altered drought tolerance phenotype” refers to detectable change inthe ability of a genetically modified plant to withstand low-waterconditions compared to the similar, but non-modified plant. In general,improved (increased) drought tolerance phenotypes (i.e., ability to aplant to survive in low-water conditions that would normally bedeleterious to a plant) are of interest.

As used herein, a “mutant” polynucleotide sequence or gene differs fromthe corresponding wild type polynucleotide sequence or gene either interms of sequence or expression, where the difference contributes to amodified plant phenotype or trait. Relative to a plant or plaint line,the term “mutant” refers to a plant or plant line which has a modifiedplant phenotype or trait, where the modified phenotype or trait isassociated with the modified expression of a wild type polynucleotidesequence or gene.

As used herein, the term “T1” refers to the generation of plants fromthe seed of T0 plants. The T1 generation is the first set of transformedplants that can be selected by application of a selection agent, e.g.,an antibiotic or herbicide, for which the transgenic plant contains thecorresponding resistance gene. The term “T2” refers to the generation ofplants by self-fertilization of the flowers of T1 plants, previouslyselected as being transgenic.

As used herein, the term “plant part” includes any plant organ ortissue, including, without limitation, seeds, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores. Plant cells can be obtained fromany plant organ or tissue and cultures prepared therefrom. The class ofplants which can be used in the methods of the present invention isgenerally as broad as the class of higher plants amenable totransformation techniques, including both monocotyledenous anddicotyledenous plants.

As used herein, “transgenic plant” includes reference to a plant thatcomprises within its genome a heterologous polynucleotide. Theheterologous polynucleotide can be either stably integrated into thegenome, or can be extra-chromosomal. Preferably, the polynucleotide ofthe present invention is stably integrated into the genome such that thepolynucleotide is passed on to successive generations. A plant cell,tissue, organ, or plant into which the heterologous polynucleotides havebeen introduced is considered “transformed”, “transfected”, or“transgenic”. Direct and indirect progeny of transformed plants or plantcells that also contain the heterologous polynucleotide are alsoconsidered transgenic.

Identification of Plants with an Improved Drought Tolerance Phenotype

We used an Arabidopsis activation tagging screen to identify theassociation between the gene encoding a lateral organ boundaries (LOB)domain protein, which we have designated “DRO5 (for Drought tolerant),”and an improved drought tolerance phenotype. Briefly, and as furtherdescribed in the Examples, a large number of Arabidopsis plants weremutated with the pSKI015 vector, which comprises a T-DNA from the Tiplasmid of Agrobacterium tumifaciens, a viral enhancer element, and aselectable marker gene (Weigel et al, supra). When the T-DNA insertsinto the genome of transformed plants, the enhancer element can causeup-regulation genes in the vicinity, generally within about 10 kilobase(kb) of the insertion. T1 plants were exposed to the selective agent inorder to specifically recover transformed plants that expressed theselectable marker and therefore harbored T-DNA insertions. Seed fromtransformed plants were planted, grown under adequate water conditions,and then deprived of water. Plants that did not wilt, and thatmaintained high water content were identified as drought tolerant.

An Arabidopsis line that showed increased drought tolerance wasidentified. The association of the DRO5 gene with the drought tolerancephenotype was discovered by analysis of the genomic DNA sequenceflanking the T-DNA insertion in the identified line. Accordingly, DRO5genes and/or polypeptides may be employed in the development ofgenetically modified plants having a modified drought tolerancephenotype (“a DRO5 phenotype”). DRO5 genes may be used in the generationof crops and/or other plant species that have improved ability tosurvive in low-water conditions. The DRO5 phenotype may further enhancethe overall health of the plant.

DRO5 Nucleic Acids and Polypeptides

Arabidopsis DRO5 nucleic acid (cDNA) sequence is provided in SEQ ID NO:1and in Genbank entry GI 30678570. The corresponding protein sequence isprovided in SEQ ID NO:2 and in GI 15236747. The TAIR designation isAt4g00220.

As used herein, the term “DRO5 polypeptide” refers to a full-length DRO5protein or a fragment, derivative (variant), or ortholog thereof that is“functionally active,” meaning that the protein fragment, derivative, orortholog exhibits one or more or the functional activities associatedwith the polypeptide of SEQ ID NO:2. In one preferred embodiment, afunctionally active DRO5 polypeptide causes an altered drought tolerancephenotype when mis-expressed in a plant. In a further preferredembodiment, mis-expression of the functionally active DRO5 polypeptidecauses improved drought tolerance. In another embodiment, a functionallyactive DRO5 polypeptide is capable of rescuing defective (includingdeficient) endogenous DRO5 activity when expressed in a plant or inplant cells; the rescuing polypeptide may be from the same or from adifferent species as that with defective activity. In anotherembodiment, a functionally active fragment of a full length DRO5polypeptide (i.e., a native polypeptide having the sequence of SEQ IDNO:2 or a naturally occurring ortholog thereof) retains one of more ofthe biological properties associated with the full-length DRO5polypeptide, such as signaling activity, binding activity, catalyticactivity, or cellular or extra-cellular localizing activity. A DRO5fragment preferably comprises a DRO5 domain, such as a C- or N-terminalor catalytic domain, among others, and preferably comprises at least 10,preferably at least 20, more preferably at least 25, and most preferablyat least 50 contiguous amino acids of a DRO5 protein. Functional domainscan be identified using the PFAM program (Bateman A et al., NucleicAcids Res (1999) 27:260-262; website at pfam.wust1.edu). A preferredDRO5 fragment comprises a DUF260 domain (PF03195). PFAM analysisidentified a DUF260 domain at amino acids 17 to 118 of SEQ ID NO:2.Functionally active variants of full-length DRO5 polypeptides orfragments thereof include polypeptides with amino acid insertions,deletions, or substitutions that retain one of more of the biologicalproperties associated with the full-length DRO5 polypeptide. In somecases, variants are generated that change the post-translationalprocessing of a DRO5 polypeptide. For instance, variants may havealtered protein transport or protein localization characteristics oraltered protein half-life compared to the native polypeptide.

As used herein, the term “DRO5 nucleic acid” encompasses nucleic acidswith the sequence provided in or complementary to the sequence providedin SEQ ID NO: 1, as well as functionally active fragments, derivatives,or orthologs thereof. A DRO5 nucleic acid of this invention may be DNA,derived from genomic DNA or cDNA, or RNA.

In one embodiment, a functionally active DRO5 nucleic acid encodes or iscomplementary to a nucleic acid that encodes a functionally active DRO5polypeptide. Included within this definition is genomic DNA that servesas a template for a primary RNA transcript (i.e., an mRNA precursor)that requires processing, such as splicing, before encoding thefunctionally active DRO5 polypeptide. A DRO5 nucleic acid can includeother non-coding sequences, which may or may not be transcribed; suchsequences include 5′ and 3′ UTRs, polyadenylation signals and regulatorysequences that control gene expression, among others, as are known inthe art. Some polypeptides require processing events, such asproteolytic cleavage, covalent modification, etc., in order to becomefully active. Accordingly, functionally active nucleic acids may encodethe mature or the pre-processed DRO5 polypeptide, or an intermediateform. A DRO5 polynucleotide can also include heterologous codingsequences, for example, sequences that encode a marker included tofacilitate the purification of the fused polypeptide, or atransformation marker.

In another embodiment, a functionally active DRO5 nucleic acid iscapable of being used in the generation of loss-of-function DRO5phenotypes, for instance, via antisense suppression, co-suppression,etc.

In one preferred embodiment, a DRO5 nucleic acid used in the methods ofthis invention comprises a nucleic acid sequence that encodes or iscomplementary to a sequence that encodes a DRO5 polypeptide having atleast 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identityto the polypeptide sequence presented in SEQ ID NO:2.

In another embodiment a DRO5 polypeptide of the invention comprises apolypeptide sequence with at least 50% or 60% identity to the DRO5polypeptide sequence of SEQ ID NO:2, and may have at least 70%, 80%,85%, 90% or 95% or more sequence identity to the DRO5 polypeptidesequence of SEQ ID NO:2. In another embodiment, a DRO5 polypeptidecomprises a polypeptide sequence with at least 50%, 60%, 70%, 80%, 85%,90% or 95% or more sequence identity to a functionally active fragmentof the polypeptide presented in SEQ ID NO:2, such as a DUF260 domain. Inyet another embodiment, a DRO5 polypeptide comprises a polypeptidesequence with at least 50%, 60%, 70%, 80%, or 90% identity to thepolypeptide sequence of SEQ ID NO:2 over its entire length and comprisesDUF260 domain.

In another aspect, a DRO5 polynucleotide sequence is at least 50% to 60%identical over its entire length to the DRO5 nucleic acid sequencepresented as SEQ ID NO:1, or nucleic acid sequences that arecomplementary to such a DRO5 sequence, and may comprise at least 70%,80%, 85%, 90% or 95% or more sequence identity to the DRO5 sequencepresented as SEQ ID NO: 1 or a functionally active fragment thereof, orcomplementary sequences.

As used herein, “percent (%) sequence identity” with respect to aspecified subject sequence, or a specified portion thereof, is definedas the percentage of nucleotides or amino acids in the candidatederivative sequence identical with the nucleotides or amino acids in thesubject sequence (or specified portion thereof), after aligning thesequences and introducing gaps, if necessary to achieve the maximumpercent sequence identity, as generated by the program WU-BLAST-2.0a19(Altschul et al., J. Mol. Biol. (1990) 215:403-410; website atblast.wust1.edu/blast/README.html) with search parameters set to defaultvalues. The HSP S and HSP S2 parameters are dynamic values and areestablished by the program itself depending upon the composition of theparticular sequence and composition of the particular database againstwhich the sequence of interest is being searched. A “% identity value”is determined by the number of matching identical nucleotides or aminoacids divided by the sequence length for which the percent identity isbeing reported. “Percent (%) amino acid sequence similarity” isdetermined by doing the same calculation as for determining % amino acidsequence identity, but including conservative amino acid substitutionsin addition to identical amino acids in the computation. A conservativeamino acid substitution is one in which an amino acid is substituted foranother amino acid having similar properties such that the folding oractivity of the protein is not significantly affected. Aromatic aminoacids that can be substituted for each other are phenylalanine,tryptophan, and tyrosine; interchangeable hydrophobic amino acids areleucine, isoleucine, methionine, and valine; interchangeable polar aminoacids are glutamine and asparagine; interchangeable basic amino acidsare arginine, lysine and histidine; interchangeable acidic amino acidsare aspartic acid and glutamic acid; and interchangeable small aminoacids are alanine, serine, threonine, cysteine and glycine.

Derivative nucleic acid molecules of the subject nucleic acid moleculesinclude sequences that hybridize to the nucleic acid sequence of SEQ IDNO: 1. The stringency of hybridization can be controlled by temperature,ionic strength, pH, and the presence of denaturing agents such asformamide during hybridization and washing. Conditions routinely usedare well known (see, e.g., Current Protocol in Molecular Biology, Vol.1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et al.,supra). In some embodiments, a nucleic acid molecule of the invention iscapable of hybridizing to a nucleic acid molecule containing thenucleotide sequence of SEQ ID NO: 1 under stringent hybridizationconditions that comprise: prehybridization of filters containing nucleicacid for 8 hours to overnight at 65° C. in a solution comprising 6×single strength citrate (SSC) (1×SSC is 0.15 M NaCl, 0.015 M Na citrate;pH 7.0), 5×Denhardt's solution, 0.05% sodium pyrophosphate and 100 μg/mlherring sperm DNA; hybridization for 18-20 hours at 65° C. in a solutioncontaining 6×SSC, 1×Denhardt's solution, 100 μg/ml yeast tRNA and 0.05%sodium pyrophosphate; and washing of filters at 65° C. for 1 h in asolution containing 0.2×SSC and 0.1% SDS (sodium dodecyl sulfate). Inother embodiments, moderately stringent hybridization conditions areused that comprise: pretreatment of filters containing nucleic acid for6 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mMTris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500μg/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40° C. ina solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA,and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hourat 55° C. in a solution containing 2×SSC and 0.1% SDS. Alternatively,low stringency conditions can be used that comprise: incubation for 8hours to overnight at 37° C. in a solution comprising 20% formamide,5×SSC, 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured sheared salmon sperm DNA;hybridization in the same buffer for 18 to 20 hours; and washing offilters in 1×SSC at about 37° C. for 1 hour.

As a result of the degeneracy of the genetic code, a number ofpolynucleotide sequences encoding a DRO5 polypeptide can be produced.For example, codons may be selected to increase the rate at whichexpression of the polypeptide occurs in a particular host species, inaccordance with the optimum codon usage dictated by the particular hostorganism (see, e.g., Nakamura Y et al, Nucleic Acids Res (1999) 27:292).Such sequence variants may be used in the methods of this invention.

The methods of the invention may use orthologs of the Arabidopsis DRO5.Methods of identifying the orthologs in other plant species are known inthe art. Normally, orthologs in different species retain the samefunction, due to presence of one or more protein motifs and/or3-dimensional structures. In evolution, when a gene duplication eventfollows speciation, a single gene in one species, such as Arabidopsis,may correspond to multiple genes (paralogs) in another. As used herein,the term “orthologs” encompasses paralogs. When sequence data isavailable for a particular plant species, orthologs are generallyidentified by sequence homology analysis, such as BLAST analysis,usually using protein bait sequences. Sequences are assigned as apotential ortholog if the best hit sequence from the forward BLASTresult retrieves the original query sequence in the reverse BLAST(Huynen M A and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen MA et al., Genome Research (2000) 10:1204-1210). Programs for multiplesequence alignment, such as CLUSTAL (Thompson J D et al, Nucleic AcidsRes (1994) 22:4673-4680) may be used to highlight conserved regionsand/or residues of orthologous proteins and to generate phylogenetictrees. In a phylogenetic tree representing multiple homologous sequencesfrom diverse species (e.g., retrieved through BLAST analysis),orthologous sequences from two species generally appear closest on thetree with respect to all other sequences from these two species.Structural threading or other analysis of protein folding (e.g., usingsoftware by ProCeryon, Biosciences, Salzburg, Austria) may also identifypotential orthologs. Nucleic acid hybridization methods may also be usedto find orthologous genes and are preferred when sequence data are notavailable. Degenerate PCR and screening of cDNA or genomic DNA librariesare common methods for finding related gene sequences and are well knownin the art (see, e.g., Sambrook, supra; Dieffenbach C and Dveksler G(Eds.) PCR Primer: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, NY, 1989). For instance, methods for generating a cDNA libraryfrom the plant species of interest and probing the library withpartially homologous gene probes are described in Sambrook et al. Ahighly conserved portion of the Arabidopsis DRO5 coding sequence may beused as a probe. DRO5 ortholog nucleic acids may hybridize to thenucleic acid of SEQ ID NO: 1 under high, moderate, or low stringencyconditions. After amplification or isolation of a segment of a putativeortholog, that segment may be cloned and sequenced by standardtechniques and utilized as a probe to isolate a complete cDNA or genomicclone. Alternatively, it is possible to initiate an EST project togenerate a database of sequence information for the plant species ofinterest. In another approach, antibodies that specifically bind knownDRO5 polypeptides are used for ortholog isolation. Western blot analysiscan determine that a DRO5 ortholog (i.e., an orthologous protein) ispresent in a crude extract of a particular plant species. Whenreactivity is observed, the sequence encoding the candidate ortholog maybe isolated by screening expression libraries representing theparticular plant species. Expression libraries can be constructed in avariety of commercially available vectors, including lambda gt11, asdescribed in Sambrook, et al., supra. Once the candidate ortholog(s) areidentified by any of these means, candidate orthologous sequence areused as bait (the “query”) for the reverse BLAST against sequences fromArabidopsis or other species in which DRO5 nucleic acid and/orpolypeptide sequences have been identified.

DRO5 nucleic acids and polypeptides may be obtained using any availablemethod. For instance, techniques for isolating cDNA or genomic DNAsequences of interest by screening DNA libraries or by using polymerasechain reaction (PCR), as previously described, are well known in theart. Alternatively, nucleic acid sequence may be synthesized. Any knownmethod, such as site directed mutagenesis (Kunkel T A et al., MethodsEnzymol. (1991) 204:125-39), may be used to introduce desired changesinto a cloned nucleic acid.

In general, the methods of the invention involve incorporating thedesired form of the DRO5 nucleic acid into a plant expression vector fortransformation of in plant cells, and the DRO5 polypeptide is expressedin the host plant.

An isolated DRO5 nucleic acid molecule is other than in the form orsetting in which it is found in nature and is identified and separatedfrom least one contaminant nucleic acid molecule with which it isordinarily associated in the natural source of the DRO5 nucleic acid.However, an isolated DRO5 nucleic acid molecule includes DRO5 nucleicacid molecules contained in cells that ordinarily express DRO5 where,for example, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

Generation of Genetically Modified Plants with a Drought TolerancePhenotype

DRO5 nucleic acids and polypeptides may be used in the generation ofgenetically modified plants having a modified, preferably an improveddrought tolerance phenotype. Such plants may further display increasedtolerance to other abiotic stresses, particular salt-stress andfreezing, as responses to these stresses and drought stress are mediatedby ABA (Thomashow, 1999 Annu. Revl Plant Physiol. Plant Mol. Biol 50:571; Cushman and Bohnert, 2000, Curr. Opin. Plant Biol. 3: 117; Kang etal. 2002, Plant Cell 14:343-357; Quesada et al. 2000, Genetics 154: 421;Kasuga et al. 1999, Nature Biotech. 17: 287-291).

The methods described herein are generally applicable to all plants.Drought tolerance is an important trait in almost any agricultural crop;most major agricultural crops, including corn, wheat, soybeans, cotton,alfalfa, sugar beets, onions, tomatoes, and beans, are susceptible todrought stress. Although activation tagging and gene identification arecarried out in Arabidopsis, the DRO5 gene (or an ortholog, variant orfragment thereof) may be expressed in any type of plant. The inventionmay directed to fruit- and vegetable-bearing plants, plants used in thecut flower industry, grain-producing plants, oil-producing plants,nut-producing plants, crops including corn (Zea mays), soybean (Glycinemax) cotton (Gossypium), tomato (Lycopersicum esculentum), alfalfa(Medicago sativa), flax (Linum usitatissimum), tobacco (Nicotiana), andturfgrass (Poaceae family), and other forage crops, among others.

The skilled artisan will recognize that a wide variety of transformationtechniques exist in the art, and new techniques are continually becomingavailable. Any technique that is suitable for the target host plant canbe employed within the scope of the present invention. For example, theconstructs can be introduced in a variety of forms including, but notlimited to as a strand of DNA, in a plasmid, or in an artificialchromosome. The introduction of the constructs into the target plantcells can be accomplished by a variety of techniques, including, but notlimited to Agrobacterium-mediated transformation, electroporation,microinjection, microprojectile bombardment calcium-phosphate-DNAco-precipitation or liposome-mediated transformation of a heterologousnucleic acid. The transformation of the plant is preferably permanent,i.e. by integration of the introduced expression constructs into thehost plant genome, so that the introduced constructs are passed ontosuccessive plant generations. Depending upon the intended use, aheterologous nucleic acid construct comprising an DRO5 polynucleotidemay encode the entire protein or a biologically active portion thereof.

In one embodiment, binary Ti-based vector systems may be used totransfer polynucleotides. Standard Agrobacterium binary vectors areknown to those of skill in the art, and many are commercially available(e.g., pBI121 Clontech Laboratories, Palo Alto, Calif.).

The optimal procedure for transformation of plants with Agrobacteriumvectors will vary with the type of plant being transformed. Exemplarymethods for Agrobacterium-mediated transformation include transformationof explants of hypocotyl, shoot tip, stem or leaf tissue, derived fromsterile seedlings and/or plantlets. Such transformed plants may bereproduced sexually, or by cell or tissue culture. Agrobacteriumtransformation has been previously described for a large number ofdifferent types of plants and methods for such transformation may befound in the scientific literature.

Expression (including transcription and translation) of DRO5 may beregulated with respect to the level of expression, the tissue type(s)where expression takes place and/or developmental stage of expression. Anumber of heterologous regulatory sequences (e.g., promoters andenhancers) are available for controlling the expression of a DRO5nucleic acid. These include constitutive, inducible and regulatablepromoters, as well as promoters and enhancers that control expression ina tissue- or temporal-specific manner. Exemplary constitutive promotersinclude the raspberry E4 promoter (U.S. Pat. Nos. 5,783,393 and5,783,394), the 35S CaMV (Jones J D et al, (1992) Transgenic Res1:285-297), the CsVMV promoter (Verdaguer B et al., Plant Mol Biol(1998) 37:1055-1067) and the melon actin promoter (published PCTapplication WO0056863). Exemplary tissue-specific promoters include thetomato E4 and E8 promoters (U.S. Pat. No. 5,859,330) and the tomato 2AIIgene promoter (Van Haaren M J J et al., Plant Mol Bio (1993)21:625-640).

In one preferred embodiment, DRO5 expression is under control ofregulatory sequences from genes whose expression is associated withdrought stress. For example, when the promoter of the drought stressresponsive Arabidopsis rd29A gene was used to drive expression ofDREB1A, Arabidopsis plants were more tolerant to drought, salt andfreezing stress and did not have the stunted stature associated withplants over-expressing the DREB1A gene from the CaMV 35S promoter(Kasuga et al, 1999 Nature Biotech 17: 287). Promoters from otherArabidopsis genes that are responsive to drought stress, such as COR47(Welin et al. 1995, Plant Mol. Biol. 29: 391), KIN1 (Kurkela and Franck,1990, Plant Mol. Biol. 15: 137), RD22BP (Abe et al. 1997, Plant Cell 9,1859), ABA1 (Accession Number AAG17703), and ABA3 (Xiong et al. 2001,Plant Cell 13: 2063), could be used. Promoters from drought stressinducible genes in other species could be used also. Examples are therab17, ZmFer1 and ZmFer2 genes from maize (Bush et al, 1997 Plant J11:1285; Fobis-Loisy, 1995 Eur J Biochem 231:609), the tdi-65 gene fromtomato (Harrak, 2001 Genome 44:368), the His1 gene of tobacco (Wei andO'Connell, 1996 Plant Mol Biol 30:255), the Vupat1 gene from cowpea(Matos, 2001 FEBS Lett 491:188), and CDSP34 from Solanum tuberosum(Gillet et al, 1998 Plant J 16:257).

In yet another aspect, in some cases it may be desirable to inhibit theexpression of endogenous DRO5 in a host cell. Exemplary methods forpracticing this aspect of the invention include, but are not limited toantisense suppression (Smith, et al., Nature (1988) 334:724-726; van derKrol et al., Biotechniques (1988) 6:958-976); co-suppression (Napoli, etal, Plant Cell (1990) 2:279-289); ribozymes (PCT Publication WO97/1032S); and combinations of sense and antisense (Waterhouse, et al.,Proc. Natl. Acad. Sci. USA (1998) 95:13959-13964). Methods for thesuppression of endogenous sequences in a host cell typically employ thetranscription or transcription and translation of at least a portion ofthe sequence to be suppressed. Such sequences may be homologous tocoding as well as non-coding regions of the endogenous sequence.Antisense inhibition may use the entire cDNA sequence (Sheehy et al.,Proc. Natl. Acad. Sci. USA (1988) 85:8805-8809), a partial cDNA sequenceincluding fragments of 5′ coding sequence, (Cannon et al., Plant Molec.Biol. (1990) 15:39-47), or 3′ non-coding sequences (Ch'ng et al., Proc.Natl. Acad. Sci. USA (1989) 86:10006-10010). Cosuppression techniquesmay use the entire cDNA sequence (Napoli et al., supra; van der Krol etal., The Plant Cell (1990) 2:291-299), or a partial cDNA sequence (Smithet al., Mol. Gen. Genetics (1990) 224:477-481).

Standard molecular and genetic tests may be performed to further analyzethe association between a gene and an observed phenotype. Exemplarytechniques are described below.

1. DNA/RNA Analysis

The stage- and tissue-specific gene expression patterns in mutant versuswild-type lines may be determined, for instance, by in situhybridization. Analysis of the methylation status of the gene,especially flanking regulatory regions, may be performed. Other suitabletechniques include overexpression, ectopic expression, expression inother plant species and gene knock-out (reverse genetics, targetedknock-out, viral induced gene silencing [VIGS, see Baulcombe D, ArchVirol Suppl (1999) 15:189-201]).

In a preferred application expression profiling, generally by microarrayanalysis, is used to simultaneously measure differences or inducedchanges in the expression of many different genes. Techniques formicroarray analysis are well known in the art (Schena M et al., Science(1995) 270:467-470; Baldwin D et al., (1999) Cur Opin Plant Biol.2(2):96-103; Dangond F, Physiol Genomics (2000) 2:53-58; van Hal N L etal., J Biotechnol (2000) 78:271-280; Richmond T and Somerville S, CurrOpin Plant Biol (2000) 3:108-116). Expression profiling of individualtagged lines may be performed. Such analysis can identify other genesthat are coordinately regulated as a consequence of the overexpressionof the gene of interest, which may help to place an unknown gene in aparticular pathway.

2. Gene Product Analysis

Analysis of gene products may include recombinant protein expression,antisera production, immunolocalization, biochemical assays forcatalytic or other activity, analysis of phosphorylation status, andanalysis of interaction with other proteins via yeast two-hybrid assays.

3. Pathway Analysis

Pathway analysis may include placing a gene or gene product within aparticular biochemical, metabolic or signaling pathway based on itsmis-expression phenotype or by sequence homology with related genes.Alternatively, analysis may comprise genetic crosses with wild-typelines and other mutant lines (creating double mutants) to order the genein a pathway, or determining the effect of a mutation on expression ofdownstream “reporter” genes in a pathway.

Generation of Mutated Plants with a Drought Tolerant Phenotype

The invention further provides a method of identifying plants that havemutations in endogenous DRO5 that confer increased drought tolerance,and generating drought-tolerant progeny of these plants that are notgenetically modified. In one method, called “TILLING” (for targetinginduced local lesions in genomes), mutations are induced in the seed ofa plant of interest, for example, using EMS treatment. The resultingplants are grown and self-fertilized, and the progeny are used toprepare DNA samples. DRO5-specific PCR is used to identify whether amutated plant has a DRO5 mutation. Plants having DRO5 mutations may thenbe tested for drought tolerance, or alternatively, plants may be testedfor drought tolerance, and then DRO5-specific PCR is used to determinewhether a plant having increased drought tolerance has a mutated DRO5gene. TILLING can identify mutations that may alter the expression ofspecific genes or the activity of proteins encoded by these genes (seeColbert et al (2001) Plant Physiol 126:480-484; McCallum et al (2000)Nature Biotechnology 18:455-457).

In another method, a candidate gene/Quantitative Trait Locus (QTLs)approach can be used in a marker-assisted breeding program to identifyalleles of or mutations in the DRO5 gene or orthologs of DRO5 that mayconfer increased tolerance to drought (see Foolad et al., Theor ApplGenet. (2002) 104(6-7):945-958; Rothan et al., Theor Appl Genet (2002)105(1):145-159); Dekkers and Hospital, Nat Rev Genet. (2002) January;3(1):22-32). Thus, in a further aspect of the invention, a DRO5 nucleicacid is used to identify whether a drought-tolerant plant has a mutationin endogenous DRO5.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention. All publications cited herein are expressly incorporatedherein by reference for the purpose of describing and disclosingcompositions and methodologies that might be used in connection with theinvention. All cited patents, patent applications, and sequenceinformation in referenced websites and public databases are alsoincorporated by reference.

EXAMPLES Example 1

Generation of Plants with a DRO5 Phenotype by Transformation with anActivation Tagging Construct

Mutants were generated using the activation tagging “ACTTAG” vector,pSKI015 (GI 6537289; Weigel D et al., supra). Standard methods were usedfor the generation of Arabidopsis transgenic plants, and wereessentially as described in published application PCT WO0183697.Briefly, T0 Agrobacterium (Col-0) plants were transformed withAgrobacterium carrying the pSKI015 vector, which comprises T-DNA derivedfrom the Agrobacterium Ti plasmid, an herbicide resistance selectablemarker gene, and the 4×CaMV 35S enhancer element. Transgenic plants wereselected at the T1 generation based on herbicide resistance. T2 seed wascollected from T1 plants and stored in an indexed collection, and aportion of the T2 seed was accessed for the screen.

Approximately 18 T2 seed from each of line tested were planted in soil.The seed were stratified for 3 days at 4° C. and then grown in thegreenhouse. Plants containing the ACTTAG insert were selected. Droughtstress was imposed on the selected plants by withholding water for 21-25days. At this time the plants were beginning to bolt.

From 6 to 10 days after the initiation of drought stress, observationsof the plants were taken daily. Transgenic plants were compared to eachother and wild-type control plants. Putative drought tolerant lines wereidentified as containing at least 2 plants that remained green andviable after wild-type plants had died or as plants that contained asoil moisture content of at least 50 mV (using a Delt-T Devices HH2 SoilMoisture Meter with a ML2x Theta Probe).

After drought tolerant lines were identified, water was applied to theplants to allow them to recover. When possible, T3 seed was collectedfrom the plants. This T3 seed was then grown and the plants assessed fordrought tolerant phenotype as described above.

The drought tolerant plants were then subjected to an “excised leaftranspiration test” in which seeds were planted, stratified, and grownfor three weeks as described above. Then, either the entire rosette or asingle leaf was excised and placed on a pre-weighed plastic weigh dishand left on the bench at room temperature. The mass of the plantmaterial was recorded immediately after excision and at 30 min intervalsafterward. The mass of drought tolerant plants often decreased lessrapidly indicating that they were transpiring less rapidly.

To detect lines containing or lacking the insert, PCR analysis wasperformed using a set of DNA oligonucleotide primers; one thathybridizes to sequences in pSKI015, the other that hybridizes tosequences flanking the insert. Genotyping of individuals analyzeddrought tolerance experiments indicated that plants containing insertidentified in W000115511 were more tolerant of drought stress thanplants without the insert.

Example 2

Characterization of the T-DNA Insertion in Plants Exhibiting the AlteredDrought Tolerance Phenotype.

We performed standard molecular analyses, essentially as described inpatent application PCT WO0183697, to determine the site of the T-DNAinsertion associated with the increased drought tolerance phenotype.Briefly, genomic DNA was extracted from plants exhibiting increaseddrought tolerance. PCR, using primers specific to the pSKI015 vector,confirmed the presence of the 35S enhancer in plants from lineW000115511, and Southern blot analysis verified the genomic integrationof the ACTTAG T-DNA and showed the presence of a single T-DNA insertionin the transgenic line.

Plasmid rescue was used to recover genomic DNA flanking the T-DNAinsertion, which was then subjected to sequence-analysis.

The sequence flanking the left T-DNA border was subjected to a basicBLASTN search and/or a search of the Arabidopsis Information Resource(TAIR) database (available at the arabidopsis.org website), whichrevealed sequence identity to nucleotides 84866-85453 on Arabidopsis BACclone F6N15 (GI 3193311), mapped to chromosome 4. Sequence analysisrevealed that the T-DNA had inserted 2091 base pairs 3′ to the startcodon of At4g0022, whose nucleotide sequence is presented as SEQ ID NO:1 and GI 18404238, and which we designated DRO5.

Example 3

Analysis of Arabidopsis DRO5 Sequence

The amino acid sequence predicted from the DRO5 nucleic acid sequence ispresented in SEQ ID NO:2 and GI 15219124.

Sequence analyses were performed with BLAST (Altschul et al., 1990, J.Mol. Biol. 215:403-410) and PFAM (Bateman et al., supra), PSORT (NakaiK, and Horton P, 1999, Trends Biochem Sci 24:34-6), among others. PFAManalysis predicted a DUF260 domain (PF03195) from amino acids 17 to 118.The function of this domain is unknown, but it is found in a number ofproteins of both monocot and dicot plant species.

Orthologs of the Arabidopsis DRO5 that can be used in the practice ofthe invention are identified in Table 1 below.

TABLE 1 Score(s) (BLAST, Coordinates of Type Species GI # % ID to DRO5Clustal, etc.) protein motif (s) Protein Oryza sativa gi|18652509Identities = BLASTP Score = PF03195: 101/232 (43%), 390 (142.3 bits),domain 1 of 1, Positives = Expect = 1.4e−35, from 33 to 134: 124/232(53%) P= 1.4e−35 score 231.7, E = 1.4e−66 EST Helianthus gi|22382607Identities = TBLASTN Score = annuus 81/161 (50%), 388 (141.6 bits),Positives = Expect = 6.6e−34, 95/161 (59%), P = 6.6e−34 EST Solanumgi|13612860 Identities = TBLASTN Score = tuberosum gi|21921820 60/164(36%), 233 (87.1 bits), gi|21921821 Positives = Expect = 7.8e−20, 83/164(50%), P = 7.8e−20

The closest homologs from Arabidopsis are shown in Table 2.

TABLE 2 At2g45420 gi|17227164 Identities = BLASTP Score = PF03195:domain 1 of 1, 115/224 (51%), 459 (166.6 bits), from 37 to 138: score236.9, Positives = Expect = 1.3e−44, E = 3.8e−68 134/224 (59%) P =1.3e−44 At2g45410 gi|25354730 Identities = BLASTP Score = PF03195:domain 1 of 1, 78/195 (40%), 303 (111.7 bits), from 16 to 117: score243.8, Positives = Expect = 4.5e−28, E = 3.1e−70 102/195 (52%) P =4.5e−28 At4g00210 gi|15236744 Identities = BLASTP Score = PF03195:domain 1 of 1, 79/196 (40%), 285 (105.4 bits), from 11 to 107: score223.4, Positives = Expect = 3.6e−26, E = 4.3e−64 105/196 (53%) P =3.6e−26 At5g63090 gi|30697782 Identities = BLASTP Score = PF03195:domain 1 of 1, 66/165 (40%), 253 (94.1 bits), from 11 to 111: score232.6, Positives = Expect = 8.9e−23, E = 7.5e−67 83/165 (50%) P =8.9e−23

Example 4

Confirmation of Phenotype/Genotype Association

RT-PCR analysis showed that the DRO5 gene was specifically overexpressedin plants from the line displaying the improved drought tolerancephenotype. Specifically, RNA was extracted from tissues derived fromplants exhibiting the DRO5 phenotype and from wild type COL-0 plants.RT-PCR was performed using primers specific to the sequence presented asSEQ ID NO: 1 and to other predicted genes in the vicinity of the T-DNAinsertion. The results showed that plants displaying the DRO5 phenotypeover-expressed the mRNA for the DRO5 gene by about 500 fold compared towild type plants, indicating that the enhanced expression of the DRO5gene is correlated with the DRO5 phenotype.

1. A transgenic plant comprising a plant transformation vectorcomprising a nucleotide sequence that encodes a DRO5 polypeptidecomprising an amino acid sequence having at least 90% sequence identityto the amino acid sequence of SEQ ID NO:2, wherein said transgenic planthas increased drought tolerance relative to a control plant.
 2. Thetransgenic plant of claim 1 wherein the transformation vector comprisesa constitutive promoter that controls expression of the DRO5polypeptide.
 3. A plant part obtained from the plant of claim 1, andcomprising said plant transformation vector.
 4. A seed obtained from theplant of claim 1 and comprising said slant transformation vector.
 5. Amethod of producing increased drought tolerance in a plant, said methodcomprising: (a) introducing into progenitor cells of the plant a planttransformation vector comprising a nucleotide sequence that encodes aDRO5 polypeptide comprising an amino acid sequence having at least 90%sequence identity to the amino acid sequence of SEQ ID NO:2, therebyproducing transformed progenitor cells and (b) growing the transformedprogenitor cells to produce cells to produce a transgenic plant, whereinsaid nucleotide sequence is expressed, and said transgenic plantexhibits increased drought tolerance relative to a control plant.
 6. Aplant obtained by the method of claim
 5. 7. A plant cell obtained fromthe plant of claim 1, and comprising said plant transformation vector.